U.S. patent application number 10/160135 was filed with the patent office on 2003-09-04 for compositions and methods for the induction of retinal detachments.
Invention is credited to Karageozian, Hampar L..
Application Number | 20030165486 10/160135 |
Document ID | / |
Family ID | 22613113 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165486 |
Kind Code |
A1 |
Karageozian, Hampar L. |
September 4, 2003 |
Compositions and methods for the induction of retinal
detachments
Abstract
A method comprising administering by ocular route a dose of a
glycol ether effective to induce retinal detachment.
Inventors: |
Karageozian, Hampar L.; (San
Juan Caspistrano, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
22613113 |
Appl. No.: |
10/160135 |
Filed: |
May 31, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10160135 |
May 31, 2002 |
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PCT/US00/42455 |
Dec 1, 2000 |
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60168830 |
Dec 3, 1999 |
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Current U.S.
Class: |
424/94.61 ;
424/731; 514/558; 514/723 |
Current CPC
Class: |
A61K 31/045 20130101;
A61K 31/77 20130101; A61K 45/06 20130101; A61K 31/765 20130101;
A61K 31/191 20130101; A61K 31/198 20130101; A61K 31/08 20130101;
A61K 31/231 20130101; A61K 31/23 20130101; A61K 31/19 20130101;
A61K 31/20 20130101; A61K 31/341 20130101 |
Class at
Publication: |
424/94.61 ;
424/731; 514/723; 514/558 |
International
Class: |
A61K 038/47; A61K
031/08; A61K 035/78; A61K 031/20 |
Claims
What is claimed is:
1. Use of a glycol ether in the manufacture of a medicament to
induce retinal detachment.
2. The use of claim 1, wherein the glycol ether is selected from
the group consisting of a calcium glucorate, a calcium lactate, a
castor oil, a glyceryl monosterate, a glyceryl oleate, a glyceryl
stearate, a glycesine, a glycine, polyethylene-propylene glycol
copolymers, polyoxyethylene glycols, polyethylene glycols,
polyethylene glycol copolymers, polyoxyethylene alkyl ethers,
polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan
fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid
esters, and a stearyl alcohol.
3. The use of claim 1, further comprising the administration of an
effective dose of a vitreal humor liquefying agent.
4. The use of claim 3, wherein said vitreal humor liquefying agent
is selected from the group consisting of glycosaminoglycanases such
as hyaluronidase, hexosaminidase, endo-.beta.-glucuronidase,
keratinase, chondroitinase AC, chondroitinase B and chondroitinase
ABC; chondroitin sulfatases such as chondroitin 4 sulfatase and
chondroitin 6 sulfatase; matrix metalloproteinases such as matrix
metalloproteinase 1, matrix metalloproteinase 2, matrix
metalloproteinase 3 and matrix metalloproteinase 9; and
protein-kinases such as streptokinase and urokinase.
5. Use of a glycol ether in the manufacture of a medicament to
induce a non-surgical retinal translocation wherein the medicament
is administered in a retinal detaching dose, wherein the detached
retina is translocated from a first position to a second position,
and the detached retinal tissue is then reattached.
6. The use of claim 5, further comprising the administration of an
effective dose of a vitreal humor liquefying agent.
7. Use of a glycol ether in the manufacture of a medicament to
protect detached retinal tissue from degeneration.
8. The use of claim 7, wherein the glycol ether is selected from
the group consisting of a calcium glucorate, a calcium lactate, a
castor oil, a glyceryl monosterate, a glyceryl oleate, a glyceryl
stearate, a glycesine, a glycine, polyethylene-propylene glycol
copolymers, polyoxyethylene glycols, polyethylene glycols,
polyethylene glycol copolymers, polyoxyethylene alkyl ethers,
polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan
fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid
esters, and a stearyl alcohol.
9. A method comprising administering by ocular route a dose of a
glycol ether effective to induce retinal detachment.
10. The method of claim 9, wherein the glycol ether is selected
from the group consisting of a calcium glucorate, a calcium
lactate, a castor oil, a glyceryl monosterate, a glyceryl oleate, a
glyceryl stearate, a glycesine, a glycine, polyethylene-propylene
glycol copolymers, polyoxyethylene glycols, polyethylene glycols,
polyethylene glycol copolymers, polyoxyethylene alkyl ethers,
polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan
fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid
esters, and a stearyl alcohol.
11. The method of claim 9, further comprising the administration of
an effective dose of a vitreal humor liquefying agent.
12. The method of claim 11, wherein said vitreal humor liquefying
agent is selected from the group consisting of
glycosaminoglycanases such as hyaluronidase, hexosaminidase,
endo-.beta.-glucuronidase, keratinase, chondroitinase AC,
chondroitinase B and chondroitinase ABC; chondroitin sulfatases
such as chondroitin 4 sulfatase and chondroitin 6 sulfatase; matrix
metalloproteinases such as matrix metalloproteinase 1, matrix
metalloproteinase 2, matrix metalloproteinase 3 and matrix
metalloproteinase 9; and protein-kinases such as streptokinase and
urokinase.
13. A method comprising the steps of: administering by ocular route
a dose of a glycol ether effective to induce retinal detachment;
translocating a portion of retinal tissue from a first position to
a second position; and reattaching said portion of retinal
tissue.
14. The method of claim 13, further comprising the administration
of an effective dose of a vitreal humor liquefying agent.
15. A method of protecting a detached retina comprising
administering by ocular route a dose of a glycol ether effective to
protect photoreceptor cells in a segment of a detached retina.
16. The method of claim 15, wherein the glycol ether is selected
from the group consisting of a calcium glucorate, a calcium
lactate, a castor oil, a glyceryl monosterate, a glyceryl oleate, a
glyceryl stearate, a glycesine, a glycine, polyethylene-propylene
glycol copolymers, polyoxyethylene glycols, polyethylene glycols,
polyethylene glycol copolymers, polyoxyethylene alkyl ethers,
polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan
fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid
esters, and a stearyl alcohol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation PCT Patent Application
Serial Number PCTUS00/42455, filed on Dec. 1, 2000, and published
in English, which claims priority to U.S. Provisional Application
Serial No. 60/168,830, filed Dec. 3, 1999, both of which are
incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods for the
non-surgical induction and treatment of retinal detachments. One
embodiment relates to a pharmaceutical composition comprising a
glycol ether effective for inducing and treating retinal
detachments. Another embodiment relates to vision restoration
achieved by surgically translocating retinal tissue. Yet another
embodiment relates to the treatment of retinal detachments.
BACKGROUND OF THE INVENTION
[0003] Retinal detachment is generally regarded as a negative
ophthalmic event to be avoided. Nevertheless, retinal detachment
has recently been used as a method with which to treat retinal
damaged, such as macular degeneration. In macular degeneration,
photoreceptor cells that allow detailed vision degenerate in one
focal area of the macula. The formation of this macular damage to
photoreceptors results in a loss of sight in the afflicted
subject.
[0004] One procedure to treat this disease is to surgically induce
a retinal detachment. In one form of this procedure, a nick is
surgically introduced into the retina of a subject. Subsequently a
small amount of fluid is then introduced between the retina and the
retinal space. The introduction of this fluid causes the separation
of the retina from the retinal pigment epithelium. Once the retina
is separated from its support layer it is then manually moved into
a new position of the retina where photoreceptor cells are healthy
and allow detailed vision, and is then reattached. Unfortunately,
there are a number of complications with this procedure.
[0005] One complication resulting from the surgical detachment of
the retina is that retinal pigment epithelia cells (RPE cells) are
released into the vitreous humor. The introduction of these cells
into the vitreous humor can lead to retinal damage by causing
proliferative retinopathy and subretinal fibrosis.
[0006] Another complication arises during the manipulation of the
detached retinal tissue as it is moved into a desired position. The
retinal tissue is extremely fragile and surgical manipulations
often will result in unintended tears in the retina. These tears
reduce any therapeutic advantages that might result from the
translocated retinal tissue.
[0007] In view of these limitations, what is needed is a
non-surgical method of inducing retinal detachment.
SUMMARY OF THE INVENTION
[0008] The invention disclosed herein is directed to compositions
and methods for inducing and treating retinal detachment. One
embodiment of the invention relates to the use of a glycol ether in
the manufacture of a medicament to induce retinal detachment.
Another embodiment relates to the use of a glycol ether in the
manufacture of a medicament to induce a non-surgical retinal
translocation wherein the medicament is administered in a retinal
detaching dose, wherein the detached retina is translocated from a
first position to a second position, and the detached retinal
tissue is then reattached. Another embodiment relates to the use of
a glycol ether in the manufacture of a medicament to protect
detached retinal tissue from degeneration. Another embodiment of
the invention contemplates a method comprising administering by
ocular route a dose of a glycol ether effective to induce retinal
detachment. Another embodiment of the invention contemplates a
method comprising the steps of administering by ocular route a dose
of a glycol ether effective to induce retinal detachment;
translocating a portion of retinal tissue from a first position to
a second position; and reattaching said portion of retinal
tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows a detached vitreous gel that has caused a
retinal tear by exerting traction upon the retina at the site of
vitreoretinal adhesion. The downward arrow indicates pressure
exerted by the detached vitreous gel. The tissue immediately above
the downward arrow shows a site of abnormal vitreoretinal adhesion.
Immediately to the right of this site of abnormal vitreoretinal
adhesion is a subretinal space. The upward facing arrow shows the
direction of liquid passing into the subretinal space. The area
below and to the right of the of subretinal space is liquid
vitreous posterior to the vitreous gel. The vitreous gel is located
immediately to the left of the liquid vitreous posterior to the
vitreous gel.
[0010] FIG. 2 illustrates two retinal detachments. FIG. 2a shows a
cross-section of an eye where the curved structure is the retina
and the rectangular structure protruding to the right represents
the vitreous gel. In the center of this figure is presented a
retinal tear. An anterior flap is indicated by the flap of tissue
extended out of the plane of the figure. The arrow indicates the
outflow of retinal components resulting from the loss of integrity
of the retina at the site of the tear. FIG. 2b also shows a retinal
tear, however, in this figure, retinal integrity is lost due to the
formation of a retinal hole that is shown on the retina itself. A
free operculum is shown above the retinal hole on the vitreous gel
that protrudes from the retina.
[0011] FIG. 3 graphically illustrates the condition of posterior
vitreal detachment. This figure shows a fluid filled central lacuna
in the vitreous humor of an eye. The arrows show fluid flow from
the central lacuna into a posterior vitreous surface separated from
the retina.
[0012] FIG. 4 is a photograph of a bisected globe in which the
cortical vitreous has partially separated from the retina.
[0013] FIG. 5 illustrates vitreoretinal traction caused by eye
movements. The direction of eye rotation is indicated by the arrow
facing counter-clockwise. The clock-wise arrow indicates the
direction of vitreoretinal drag and also points toward an
accumulation of subretinal fluid. Immediately above the subretinal
fluid is the vitreous gel.
[0014] FIG. 6 illustrates the extension of retinal detachment
associated with eye movements. The direction of eye rotation is
indicated by the arrow facing counter-clockwise at the top of the
figure. The arrows at the bottom of the figure facing clockwise
indicate liquid pushing against vitreous gel adjacent to a retinal
tear. Fluid is pushed through the retinal tear, as indicated by the
two-headed clockwise facing arrow. The arrow pointing toward the
top of the figure indicates liquid currents pushing against the
vitreous gel.
[0015] FIG. 7 illustrates the extension of subretinal fluid
associated with eye movements. The direction of eye rotation is
indicated by the arrow facing counter-clockwise. Subretinal fluid
causes extended retinal detachment, as indicated by the downward
facing arrow.
[0016] FIG. 8 illustrates superotemporal rhegmatogenous retinal
detachments. In the top figure is shown a retinal detachment. The
tear is at the top and center of the figure. An area of retinal
detachment is shown in the upper left quadrant of the circular
retina illustrated. In the bottom figure, the macular is shown by a
darkened oval to the right of the center of the figure. The area of
retinal detachment is shown in the bottom left quadrant of the
graph.
[0017] FIG. 9 illustrates a retinal detachment involving two
quadrants of a retina. The area of retinal break is shown by the
two-headed arrow.
[0018] FIG. 10 illustrates a retinal detachment involving the lower
quadrants of a globe. The area of retinal break is shown by the two
two-headed arrows.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The invention disclosed herein relates to compositions and
methods for the induction of temporary retinal detachment in the
eye of a subject mammal without retinal surgery. Additionally,
treatment of retinal detachments is also provided below. To achieve
these goals, typically a short-chain polymeric alcohol, preferably
polyethylene glycol or another retinal detaching agent, is
administered to the vitreal humor of a subject in need of retinal
relocation therapy.
[0020] Retinal Adhesion
[0021] To better understand the problem to be solved, it will be
helpful to understand the structure of the retina and the molecules
and structures responsible for retinal adhesion. One important
structure is the interphotoreceptor matrix (IPM). The IPM is not
simply a sticky glue. It contains complex molecules, such as
glycosaminoglycans, and has an elaborate structure in which domains
of distinct chemical characteristics surround the rods and cones.
These can be demonstrated by staining the matrix material with
fluorescent binding molecules. The matrix serves several functions,
which include physical support of the photoreceptors, transfer of
nutrients and visual pigments, and the formation of an adhesive
bond between neural retinal and RPE. These functions are largely
controlled by the RPE, not only through synthesis of matrix
materials and transport proteins, but also acutely through the
transport of ions and water. The degree to which the IPM is
hydrated or dehydrated alters its bonding properties and
viscosity.
[0022] Retinal adhesion is a complex process, involving several
complementary and interactive mechanisms. The neural retina is
pressed in place by the vitreous gel, intraocular fluid pressure,
and RPE water transport, which drive water through the semi
permeable tissue. Also, some physical resistance prevents
separation of the outer segments from the enveloping RPE
microvilli. However, the strongest mechanism for the bonding of the
retina to RPE space appears to be the IPM. When the neural retina
is freshly peeled from the RPE, the IPM material stretches
dramatically before it breaks, which shows that it is firmly
attached to both neural retinal and RPE surfaces. It also is
important to recognize that, despite these physical forces of
adhesion, the strength of neural retinal adhesion is constantly and
acutely dependent upon vital metabolism. .sup.2For example, neural
retinal adhesive force drops to near zero within minutes after
death and adhesive strength can be reversibly restored or enhanced
by tissue oxygenation. The likely basis of these metabolic effects
is water transport across the RPE, which controls the hydration and
local ionic environment in the subneural retinal space and thereby
the boding properties of the IPM material.
[0023] Neural retinas do not detach easily, which is perhaps a
reflection of these multiple mechanisms for keeping it in place.
However, detachment is more frequent in older eyes (which may be
metabolically less competent); serious neural retinal detachments
often are associated with local ischemic conditions, such as
eclampsia and severe hypertension. When neural retinas have been
detached experimentally and then allowed to reattach, full adhesive
strength is not regained for more than 1 month. .sup.3Resynthesis
of matrix domains after enzymatic destruction requires about 2
weeks and additional time may be needed for the RPE and
photoreceptors to regain full microvillous intercalation. The
clinical message is that neural retinal attachment is a complex and
metabolically vital process, which is relevant to the
pathophysiology of both neural retinal detachment, and the process
of repair.
[0024] .sup.4Retinal attachment usually is maintained by: an
adhesive-like mucopolysaccharide in the subretinal space; entotic
pressure differences between the choroid and subretinal space;
hydrostatic or hydraulic forces related to intraocular pressure;
and metabolic transfer of ions and fluid by the retinal pigment
epithelium (RPE).
[0025] Retinal detachment occurs when the combination of factors
that promote retinal detachment overwhelms the normal attachment
forces.
[0026] Rhegmatogenous retinal detachments are an important
potential cause of reduced visual acuity, particularly in the
subgroup of individuals who are predisposed to the development of
retinal tears. Nearly all symptomatic rhegmatogenous retinal
detachments progress to total blindness unless they are repaired
successfully. Timely recognition of the symptoms and signs of
retinal detachment is important to maximize the chances of a
favorable surgical outcome and preserve visual acuity.
[0027] Epidemiology and Pathogenesis.sup.4
[0028] The essential requirements for a rhegmatogenous retinal
detachment include a neural retinal break (rhegina=rent or rupture)
and vitreous liquefaction sufficient to allow vitreous fluid to
pass through the break into the subretinal space. The usual
pathologic sequence that results in retinal detachment is vitreous
liquefaction followed by a posterior vitreous detachment (PVD),
which in turn causes a retinal tear at the site of a significant
vitreoretinal adhesion (FIG. 1). All ocular conditions that are
associated with an increased prevalence of vitreous liquefaction
and PVD or with an increased number or extent of vitreoretinal
adhesion are associated with a high incidence of retinal
detachment.
[0029] Factors That Cause Retinal Detachment
[0030] The major factors associated with the development of retinal
detachment include retinal breaks, vitreous liquefaction and
detachment, traction on the retina (vitreoretinal traction), and
intraocular fluid currents associated with movement of liquid
vitreous and subretinal fluid. The majority of eyes with retinal
breaks do not develop retinal detachment because the physiologic
forces present are sufficient to hold the retina in place.
[0031] Retinal Breaks
[0032] Retinal breaks traditionally are classified as holes, tears
or dialyses. Retinal holes are fall-thickness retinal defects that
are not associated with persistent vitreoretinal traction in their
vicinity. They occur usually as a result of localized atrophic
intraretinal abnormalities.
[0033] Retinal tears usually are produced by PVD and subsequent
vitreoretinal traction at the site of a significant vitreoretinal
adhesion (FIG. 1 and FIG. 2). Vitreous traction usually persists at
the edge of a tear, which promotes progression of the retinal
detachment.
[0034] Dialyses are linear retinal breaks that occur along the ora
serrata. While most are strongly associated with blunt ocular
trauma, dialyses can occur spontaneously in certain
individuals.
[0035] Vitreous Liquefaction and Detachment
[0036] Aging of the human vitreous (synchysis senilis) is
characterized by liquefaction of the vitreous gel and the
occurrence of progressively enlarging pools of fluid (lacunae)
within the gel. These optically empty liquid spaces continue to
coalesce as age advances; extensive liquefaction within the
vitreous cavity leads to a reduction in both the shock-absorbing
capabilities and the stability of the gel. Accelerated vitreous
liquefaction is associated with significant myopia, surgical and
nonsurgical trauma, intraocular inflammation, and a variety of
other congenital, inherited, or acquired ocular disorders.
[0037] Posterior vitreous detachment, routinely termed PVD or
posterior vitreous detachment, usually occurs as an acute event
after significant liquefaction of the vitreous gel. The
precipitating event probably is a break in the posterior cortical
vitreous in the region of the macula. .sup.5This is followed by the
immediate passage of intravitreal fluid into the space between the
cortical vitreous and retina (FIG. 3). Characteristically, this
rapid movement of fluid and the associated collapse of the
remaining structure of the gel result in extensive separation of
the vitreous gel and retina posterior to the vitreous base,
especially in the superior quadrants. Partial PVDs usually progress
rapidly (within days) to become complete (FIG. 4).
[0038] Traction on the Retina
[0039] Vitreoretinal traction has a number of causes, which range
form simple action of gravitational force on the vitreous gel to
prominent transvitreal fibrocellular membranes. Gravitational force
is important and probably accounts for the high percentage of
superior retinal tears (80%). However, rotational eye movements,
which exert strong forces on all vitreoretinal adhesions, probably
are more important causes or ongoing vitreoretinal traction.sup.6.
When the eye rotates, the inertia of the detached vitreous gel
causes it to lag behind the rotation of the eyewall and, therefore,
the attached retina. The retina at the site of vitreoretinal
adhesion exerts force on the vitreous gel, which causes the
adjacent vitreous to rotate. The vitreous gel, because of its
inertia, exerts an equal and opposite force on the retina, which
can cause a retinal break or separate the neural retina further
form the pigment epithelium if subretinal fluid is already present
(FIG. 5). When the rotational eye movement stops, the vitreous gel
continues its internal movement and exerts vitreoretinal traction
in the opposite direction.
[0040] In addition to gravitational and inertial forces,
vitreoretinal traction can be caused by contracture of intraocular
fibroproliferative tissue associated with trauma, retinal vascular
proliferative disorders, and other conditions. This type of
traction does not always create a retinal break. Instead, a
traction retinal detachment may be produced. There are classic
features that often are used to differentiate this type of
detachment form the rhegmatogenous variety.sup.7. Sometimes
significant vitreoretinal traction initially causes a localized
traction detachment, which later becomes more extensive with the
development of a retinal break.
[0041] Liquid Currents
[0042] Continuous flow of liquid vitreous through a retinal break
into the subretinal space is necessary to maintain a rhegmatogenous
retinal detachment, because subretinal fluid is absorbed
continually from the subretinal space via the RPE. This flow is
encouraged by vitreoretinal traction, which tends to elevate the
retina from the RPE. Rotary eye movements cause liquid currents in
the vitreous to push against the gel adjacent to the retinal break
and to dissect beneath the edge of retinal break into the
subretinal space (FIG. 6). Subsequent eye movements also have an
inertia effect on the subretinal fluid that favors extension of the
retinal detachment (FIG. 7).
[0043] Conditions that Predispose an Eye to Retinal Detachment
[0044] Retinal detachments are relatively unusual in the general
population the accepted annual incidence FIG. 1 approximately
1:10,0008. However, a variety of ocular and systemic disorders are
associated with pathologic vitreous liquefaction, premature
vitreous detachment, and extensive sites of vitreoretinal adhesion.
These conditions, therefore, also are associated with increased
chances of retinal detachment. Particularly important predisposing
entities include high myopia, pseudophakia and aphakia, blunt and
penetrating ocular trauma, and cytomegalovirus retinitis associated
with acquired immunodeficiency syndrome.
[0045] Although cataract surgery has been performed on only
approximately 3% of the general population, up to 40% of eyes with
retinal detachment have had prior cataract surgery.sup.9. Retinal
detachment represents the most significant potential post surgical
complication of cataract surgery, as it occurs in nearly 1% of
pseudophakic eyes.sup.10. Removal of the natural lens is believed
to increase the risk of retinal detachment because of its effect on
vitreous liquefaction and subsequent premature PVD.sup.11. The
status of the posterior capsule determines the rapidity of vitreous
liquefaction. It is clear that opening the posterior capsule,
either surgically or with a neodymium:yttruim-aluminum-garnet
laser, significantly increases the incidence of retinal
detachment.sup.12.
[0046] High myopia (>6.0D myopia) is associated with at least a
threefold increased incidence of retinal detachment.sup.13. Severe
ocular trauma is believed to be responsible for 10-15% of retinal
detachments, and up to 50% of patients who have a diagnosis of
cytomegalovirus retinitis develop a rhegmatogenous retinal
detachment within 1 year.sup.14.
[0047] Risk factors for retinal detachment are not mutually
exclusive and may be additive. For example, prior cataract
extraction and nonsurgical trauma are more likely to be complicated
by retinal detachment in myopic eyes. Pathologic vitreoretinal
changes often occur bilaterally--patients who have a retinal
detachment in one eye usually have a substantially increased risk
of retinal detachment in the fellow eye, provided that additional
acquired risk factors are comparable.
[0048] The early symptoms of acute retinal detachment are the same
as those of acute posterior vitreous detachment (PVD)--the sudden
onset of tiny dark floating objects, frequently associated with
photopsia (flashes). Photopsia flashes are usually brief, in the
temporal visual field, and are best seen in the dark immediately
following eye movement. Loss of visual field does not occur until
sufficient fluid has passed through the retinal break(s) to cause a
retinal detachment posterior to the equator. Retinal detachments
with a relatively small amount of subretinal fluid (less than two
disk diameters from the break) often are not accompanied by visual
field loss; these are termed subclinical detachments. Rarely, but
especially in young female myopes, asymptomatic retinal detachment
can occur. This is most common inferiorly and usually occurs as a
result of atrophic holes in lattice degeneration.sup.15.
[0049] The vast majority of retinal breaks are located at the
equator or more interiorly; subretinal fluid initially accumulates
in the retinal periphery, where it causes a corresponding loss of
peripheral vision in the area that is related inversely to the
location of the retinal detachment (FIG. 8). The loss of peripheral
vision (a `curtain effect`) increases as the detachment enlarges;
central visual acuity is lost when subretinal fluid passes beneath
the macula. Frequently, patients do not notice any symptoms until
the macula becomes involved.
[0050] Retinal breaks associated with small amounts of subretinal
fluid are difficult to detect; however, the diagnosis becomes more
obvious as the retinal detachment increases in size. A stereoscopic
vitreoretinal examination typically reveals an elevated sensory
retina in the arc of detachment, but the critically important
identification of all retinal breaks may remain difficult--it is
considerable easier to diagnose the retinal detachment than to
detect all retinal breaks.
[0051] The effects of gravity mean that the topography of a retinal
detachment is of major value in the prediction of the most likely
locations of retinal breaks.sup.16. Retinal breaks usually are
present superiorly within the area of detachment. Thus, if a
retinal detachment involves one upper quadrant or both the superior
and inferior quadrants on one side of the vertical meridian, the
responsible retinal break is likely to be near the superior edge of
the detachment (FIG. 9). Retinal detachments that involve the
inferior quadrants tend to follow the same rules, but the
progression of the detachment often is much slower, and symmetric
spread of subretinal fluid may occur on both sides of the break.
Therefore, detachments that involve on or both inferior quadrants
may have a break near the superior margin of the detachment or in
the meridian that bisects the area of detachment (FIG. 10).
Nevertheless, since multiple retinal breaks are common, the entire
periphery of the detached retina must be meticulously examined.
[0052] Repair and Regeneration
[0053] Although of neural origin, the retinal pigment epithelium
(RPE) can be a plenipotential tissue. In amphibians, RPE cells can
regenerate lens, neural retina, and other components of eye, this
does not take place in humans. Nevertheless, the RPE is capable of
local repair (unlike the neural retina) and cells may migrate and
take on altered characteristics. After a laser bum, for example,
the RPE cells that surround the burn begin to divide and small
cells fill the defect to form a new blood--retinal barrier within
1-2 weeks.sup.18. In degenerative disease, such as retinitis
pigmentosa, RPE cells migrate into the injured neural retina and
sometimes come to rest around vessels to contribute to the
characteristic bone spicule appearance. An overly vigorous RPE
response can lead to duplicated layers of RPE cells and RPE
scarring, which may be a part of a macular degenerative process. In
the extreme, RPE cells contribute to proliferative
vitreoretinopathy. Growth factors form the RPE may, at times, help
contain unwanted proliferation, and at other times stimulate
vascular or fibrous growth. Functionally, the most useful RPE
repair characteristic is the ability to heal defects. The value to
photocoagulation for macular edema and proliferative diabetic
retinopathy may, in part, depend on the ability of RPE cells to
seal laser scars, re-establish a degree of normal transport, and
avoid unnecessary leakage of proteins into the subneural retinal
space.
[0054] As illustrated by the discussion above, retinal detachments
generally have negative connotations for the sight of the afflicted
subject. Recently, however, it has been theorized that by
intentionally detaching some or all of a subject's retina, and the
relocating or reorienting the retina into a new configuration, can
have certain positive ophthalmic results. In fact, retinal
relocation therapy is contemplated to be efficacious in the
treatment of a number of different conditions such as retinal
diseases (retinopathy) including central serous retinopathy,
diabetic retinopathy, ocular ischemic syndrome, photic retinopathy,
bardet-biedl syndrome, post-infections, proliferative, purtscher's,
radiation retinopathy, neovascularization of the angle, venous
stasis, chorioretinopathy, retinopathy of prematurity, and
retinopexy are all contemplated for treatment with the methods and
compounds described herein.
[0055] A major problem with retinal relocation therapy is that the
retina is difficult to detach and relocate without causing
unintended complications to the retinal space of the treated eye.
Additionally, physical manipulation of a detached retina often
results in degradation of the detached retinal tissue, which in
turn reduces the efficacy of the retinal relocation therapy. The
methods of the invention disclosed herein solve these problems. In
the presence of a short chain polymeric alcohol, such as
polyethylene glycol (PEG), or another retinal detachment agent, the
retina of a subject is induced to detach from the retinal matrix
holding the retina on the eye without damaging the eye or the
detached retinal tissue.
[0056] Without being bound to any particular theory, the detachment
of retinal tissue from a subject's eye has been induced by the
application of short chain polymeric alcohols, such as polyethylene
glycol (PEG). The use of such compounds permits the selective
detachment of a retina in a subject. Once detached, the retina can
be relocated to a second position at which retinal reattachment can
occur.
[0057] The methods of the invention disclosed herein are capable of
providing a number of advantages over traditional surgical methods
of retinal detachment. For example, the incidence of cell release
into the vitreal space is greatly reduced when retinal detachment
is induced using the methods disclosed herein. Also, the integrity
of the retinal tissue following reattachment is substantially less
impaired when the non-surgical detachment method of the invention
disclosed herein is used.
[0058] As discussed above, the vitreous humor is attached to the
retina in several locations. Vitreoretinal traction resulting from
these interaction often produces tears in detached retinal tissue.
Accordingly, reduction or removal vitreoretinal traction will help
to preserve the integrity of a detached retina. In one embodiment,
retinal integrity is protected following detachment by
administering hyaluronidase to liquefy the vitreous humor. Vitreal
liquefaction is taught in U.S. Pat. No. 5,866,120, which is hereby
incorporated by reference.
[0059] A Preferred Hyaluronidase Preparation for Ophthalmic
Administration
[0060] A general formulation for an injectable, thimerosal free
hyaluronidase preparation of the present invention is shown in
Table 1 and Table 2 as follows.
1TABLE 1 General Formulation Ingredient Quantity Hyaluronidase ACS
Up to 8000 International Units Lactose USP 5.0 mg-130.0 mg
Phosphate USP 0.01-100 mmoles
[0061] These formulation ingredients are initially dissolved in
sterile water, sterile filtered and subsequently lyophilized to a
dry composition. The lyophilized composition is packaged for
subsequent reconstitution prior to use; in a suitable solvent such
as sterile isotonic saline solution or balanced salt solution.
2TABLE 2 Liquid Formulation Ingredient Quantity (w/v) Liquid
Formulation in a Sterile 3.0 ml Glass Vial-0.3 ml Fill Volume
Hyaluronidase ACS 1500 I.U./ml Lactose USP 1.25 mg/ml Potassium
Phosphate Monobasic USP 0.305 mg/ml Potassium Phosphate Dibasic USP
0.48 mg/ml Sodium Chloride USP 9.0 mg/ml Water for Injection USP
Q.S. Liquid Formulation in a Sterile Pre-filled Syringe-0.15 ml
Fill Volume Hyaluronidase ACS 1500 I.U./ml Lactose USP 1.25 mg/ml
Potassium Phosphate Monobasic USP 0.305 mg/ml Potassium Phosphate
Dibasic USP 0.48 mg/ml Sodium Chloride USP 9.0 mg/ml Water for
Injection USP Q.S.
[0062] The biochemical method for inducing a retinal detachment in
the eye of a subject mammal without retinal surgery is achieved by
injecting into the subject vitreous a glycol ether or a glycol
ether derivative. In this regard, applicant has devised a method
for a non-surgical retinal detachment, said method further
comprising the step of contracting the vitreous with at least one
enzyme in an amount, which is active to accelerate the liquefaction
of the vitreous. This vitreous liquefaction of the present
invention may be performed without any other surgical manipulation
or vitrectomy, thereby avoiding the potential risks and
complications associated with vitrectomy.
[0063] Specific glycosaminoglycanase enzymes that exhibit this
vitreous liquefying effect include: hyaluronidase; hexosaminidase;
endo-.beta.-glucuronidase; keratinase; chondroitinase ac;
chondroitinase b; chondroitinase abc; and chondroitin 4 sulfatase
and chondroitin 6 sulfatase.
[0064] Specific methalloproteinase enzymes that exhibit this
vitreous liquefying effect include: matrix metalloproteinase-1;
matrix metalloproteinase-2;matrix metalloproteinase-3; and matrix
metalloproteinase-9.
[0065] Specific protein-kinase enzymes that exhibit this vitreous
liquefying effect include: streptokinase and urokinase.
[0066] Many kinds of hyaluronidase enzyme free of thimerosal
preservative can be used, however, the term "hyaluronidase ACS" as
used here describes a preferred hyaluronidase which the applicants
have determined to result in less ophthalmic toxicity than other
hyaluronidase preparations while exhibiting desirable therapeutic
efficacy in a number of ophthalmic applications.
[0067] The hyaluronidase (ACS) of the present invention, and/or the
exclusion of thimerosal form its formulation, provides a
hyaluronidase preparation which is non-toxic to the eye when
administered at dosage levels at which other hyaluronidase
preparations preserved with thimerosal would cause toxic
effects.
[0068] The induction of a retinal detachment in the present
invention is achieved by injecting glycol ether or a glycol ether
derivative into the vitreous. The said method generally is
comprised of the steps of contracting, with the vitreous humor, a
quantity of glycol ether or glycol ether derivative at a dose,
which is insufficient to accelerate the detachment of the retina
temporarily, without causing damage to the retina or other tissues
of the eye.
[0069] The methods of non-surgical retinal detachment disclosed
herein are applicable mammalian eyes. It is readily apparent to
those of skill in the art that a wide variety of mammalian
organisms could benefit from the methods disclosed herein.
[0070] Administration
[0071] Preferably the retinal detachment compounds are administered
intravitreally. In one embodiment, the route of retinal detachment
compound administration is by intraocular injection directly into
the vitreous body. Other routes of for the administration of
retinal detachment compounds are contemplated. In fact, any other
suitable route of administration that results in the distribution
of the retinal detachment compound to the vitreous body to cause
retinal detachment is encompassed by the invention disclosed
herein.
[0072] Regarding volumes of administration, one of ordinary skill
in the art would be aware that the total volume of liquid
administered to an eye is limited by the amount of pressure
generated by the administration. The volume of the composition
administered to an eye using the methods described herein is not to
exceed a volume that would create a detrimental amount of
intraocular pressure. Typically, a volume range from about 1 to 250
.mu.l is administered. Preferably a range from about 25 to 100
.mu.l is administered. More preferably, about 50 .mu.l is
administered.
[0073] Retinal Detachment Compounds
[0074] A number of retinal detachment compounds are contemplated
for use in the disclosed invention. Such compounds include, but are
not limited to short chain polymeric alcohols having 1 to 4 (e.g.,
polyethylene glycol), glycol ethers and glycol ether
derivatives.
[0075] Specific glycol ether and glycol ether derivatives that
exhibit the retinal detachment effect include: calcium glucorate;
calcium lactate; castor oil; glyceryl monosterate; glyceryl oleate;
glyceryl stearate; glycesine; glycine; polyethylene--propylene
glycol copolymers such as: poloxamer 124, 188, 237, 338, 407;
polyethylene glycol: polyoxyethylene glycol; peg 200, 300, 400,
540, 600, 900, 1000, 1450, 1540, 2000, 3000, 3350, 4000, 4600,
8000, 20000; polyethylene glycol (40) monostearate; polyethylene
glycol (50) monostearate; polyethylene glycol (400) monostearate;
polyethylene glycol (400) distearate; polyethylene glycol
trimethylnonyl ethers; peg-4 laurate; peg-6 laurate; peg-6 oleate;
peg-5 stearate; peg-8 stearate; peg 10 propylene glycol glyceryl
laurate; polyoxyethylene alkyl ethers, such as: poloxyl 20
cetostearyl ethers and brij 52, 56, 58, 30, 35, 92, 96, 98, 72, 76,
78, 700; polyoxyethylene castor oil derivatives, such as: polyoxyl
5 castor oil, p-9 castor oil, p-15 castor oil, p-40 castor oil,
p-40 hydrogenated castor oil, p-60 hydrogenated castor oil;
polyoxyethylene sorbitan fatty acid esters, such as: polysorbate
20, 21, 40, 60, 61, 65, 80, 81, 85, 120; polyoxyethylene stearates,
such as: polyoxyl 2 stearate, p-6 stearate, p-8 stearate, p-12
stearate, polyoxyl 20 stearate, p-30, p-40, p-50, p-100, p-150
stearates; polysorbate 20; sorbitan fatty acid esters, such as:
sorbitan monoisostearate; sorbitan laurate; sorbitan oleate;
sorbitan palmitate; sorbitan stearate; and stearyl alcohol.
[0076] The preferred injectable retinal detachment compound
containing solutions may contain retinal detachment compounds at
doses of about 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, or about 100% w/v. Polyethylene
Glycol 300, without causing toxic damage to the eye, along with
inactive ingredients, which cause the solution to be substantially
isotonic or hypertonic, and a pH, is suitable for injection into
the eye. This glycol ether preparation is preferably devoid of any
preservative. Such solution for injection may be in the form of a
sterile solution, or could be in a pre-filled syringe ready to be
injected into the eye.
[0077] A Preferred Glycol Ether Preparation for Ophthalmic
Administration
[0078] General formulations for injectable polyethylene glycol 300
preparations are shown in the following tables.
3TABLE 3 General Formulation Ingredient Quantity Polyethylene
Glycol 300 USP 100% w/v
[0079]
4TABLE 4 Preferred Formulation Ingredient Quantity PEG 300 USP 50%
w/v Sterile Isotonic Saline USP 50% w/v
[0080]
5TABLE 5 Preferred Formulation Ingredient Quantity Quantity PEG 300
USP 75% w/v Water for Injection USP 25% w/v
[0081] A general formulation for an injectable polyethylene glycol
400 preparation of the present invention is shown in the following
tables.
6TABLE 6 General Formulation Ingredient Quantity Polyethylene
Glycol 400 USP 100% w/v
[0082]
7TABLE 7 Preferred Formulation Ingredient Quantity PEG 400 USP 50%
w/v Sterile Isotonic Saline USP 50% w/v
[0083]
8TABLE 8 Preferred Formulation Ingredient Quantity Quantity PEG 400
USP 75% w/v Water for Injection USP 25% w/v
[0084] Suitable choices and amounts of a particular retinal
detachment compound for use in the methods described herein can
easily be determined by one of ordinary skill in the art, for
example, by performing the experiments described in Example 1 and
applying scientific methodology to test variables while running
appropriate controls.
[0085] Retinal Reattachment
[0086] A variety of methods are contemplated to reattach a detached
retina. In one embodiment of the invention, the retinas detached
using the methods of the disclosed invention spontaneously
reattach. In another embodiment, the detached retinas are
reattached using cryotherapy (cryopexy), photocoagulation, and
diathermy. Additional methods of reattachment using laser induced
scleral shrinkage and transscleral photocoagulation to facilitate
retinal reattachment are taught in U.S. Pat. No. 5,688,264, which
is hereby incorporated by reference. Retinal reattachment and
retinal tissue protection are promoted by the administration of the
compositions discussed herein.
[0087] Particular aspects of the invention may be more readily
understood by reference to the following examples, which are
intended to exemplify the invention, without limiting its scope to
the particular exemplified embodiments.
EXAMPLE 1
[0088] Determination of Suitable Retinal Detachment Compounds and
Concentrations Thereof
[0089] Twelve (12) pigmented rabbits were divided into 2 groups of
6 animals each. Group I consisted of six animals that were injected
OD with 50 .mu.l of PEG 300 at 1, 10, 20, 50, 75, and 100 % w/v. As
a control, these same animals received saline injections OS and
thus served as a control group. The effects of these injections
were monitored using standard ophthalmic methods suitable to
determine the health of the eye so injected as well as adapted to
determine the extent of retinal detachment. These methods including
light microscopy, electroretinography, and ultrasonography
techniques were used to objectively determine the presence of
retinal detachment.
EXAMPLE 2
[0090] Ophthalmic Toxicities of Thimerosal Hyaluronidase (ACS) and
Hyaluronidase (Wydase.RTM.) in Rabbits
[0091] Fifty-two (52) healthy rabbits of the New Zealand Cross
variety (26 male, 26 female) weighing 1.5 kg to 2.5 kg were
individually marked for identification and were housed individually
in suspended cages. The animals received a commercially available
pelleted rabbit feed on a daily basis, with tap water available ad
libitum.
[0092] The animals were divided into thirteen groups of 4 animals
each (2 males, 2 females). Two animals in each group (1 male, 1
female) were selected for pretreatment fundus photography and
fluorescein angiography.
[0093] The fundus photography was performed by restraining the
animals and visualizing the optic nerve, retinal arcades and fundus
with a KOWA.RTM. RC-3 Fundus Camera loaded with Kodak Gold 200 ASA
film.
[0094] The fluorescein angiography involved a 1.5-ml injection of
2% sterile fluorescein solution via the marginal ear vein.
Approximately 30 seconds post-injection the fluorescein was
visualized upon localization of the optic nerve, retinal vessels
and fundus.
[0095] The following day, each animal was anesthetized by
intravenous administration of a combination of 34 mg/kg of ketamine
hydrochloride and 5-mg/kg xylazine. The eyelids were retracted
using a lid speculum, and the eyes were disinfected with an
iodine-povidone wash.
[0096] Experimental treatments of either balance salt solution
(BSS), BSS+thimerosal, (Wydase.RTM.) or hyaluronidase (ACS) were
administered by injection using a 1 cc tuberculin syringe with a 30
gauge, 0.5 inch needle attached thereto. The hyaluronidase (ACS)
solution utilized in this example was free of thimerosal and
constituted the specific preferred hyaluronidase (ACS) formulation
set forth in Table II here above. The experimental treatments
administered to each animal group were as follows:
9TABLE 9 Group # Treatment 1 BSS 2 BSS + 0.0075 mg Thimerosal 3 BSS
+ 0.0025 mg Thimerosal 4 Hyaluronidase (Wydase) 1 IU 5
Hyaluronidase (Wydase) 15 IU 6 Hyaluronidase (Wydase) 30 IU 7
Hyaluronidase (Wydase) 50 IU 8 Hyaluronidase (Wydase) 150 IU 9
Hyaluronidase (ACS) 1 IU 10 Hyaluronidase (ACS) 15 IU 11
Hyaluronidase (ACS) 30 IU 12 Hyaluronidase (ACS) 50 IU 13
Hyaluronidase (ACS) 150 I.U.
[0097] The day following the injections (Day 1) the 26 animals,
which were subjected to the fundus photography and fluorescein
angiography, were observed using the same methods as for the
pre-dose examination.
[0098] On Day 2 following the injections, the 13 male rabbits that
had received the fundus photography and fluorescein angiography at
pre-dose and Day 1, as well as the 13 female rabbits that were not
selected for photography were euthanized with a sodium
pentobarbital drug. The eyes were then surgically removed and
placed in a fixture solution of 2.5% glutararaldehyde with 0.1M
phosphate buffered saline at pH 7.37. Alternatively, one randomly
selected rabbit was euthanized by pentobarbital injection but then
fixed by intracardiac injection of the gluteraldehyde solution into
the left ventricle to determine the effect of the fixation
procedure on the histology findings within the enucleated eyes.
[0099] On Day 7, the 13 female rabbits that had been previously
photographed and angiography performed were subjected to same
observations following the methods previously described.
[0100] The remaining 26 animals were euthanized as described above
7 days after dosing. The eyes were fixed in the same manner as
those, which had been fixed on day 2. Also, one randomly selected
rabbit was subjected to the same intracardiac gluteraldehyde
fixation procedure described here above for the previously randomly
selected animal.
[0101] The eyes of the animals treated in this example were
examined grossly and microscopically for evidence of
treatment-related toxicities.
[0102] In summary, the eyes of the BSS-treated control group were
free of toxicity at 2 and 7 days post dose.
[0103] The eyes of the Group No. 2 animals treated with
BSS+thimerosal (0.0075 mg) were free of toxicity at day 2, but
exhibited evidence that there was a breakdown of the blood retinal
barrier at day 7.
[0104] The Group No. 3 animals treated with BSS+thimerosal (0.025
mg) exhibited severe treatment-related toxic effects, at day 2 and
7 post dose.
[0105] The Group No. 4 animals treated with Wydase.RTM. at the 1 IU
dose were free of toxicity at days 2 and 7, however, the eyes of
the animals in Group Nos. 5-8 treated with Wydase.RTM. at dosages
ranging from 15 IU-150 IU exhibited generally dose-related toxic
effects at days 2 and 7 post dose.
[0106] The eyes of animals in treatment Groups Nos. 9-13 treated
with hyaluronidase (ACS) at dosages ranging from 1 IU through 150
IU, were free of evidence of toxic effects at days 2 and 7 post
dose.
EXAMPLE 3
[0107] Ophthalmic Toxicities of Thimerosal, Hyaluronidase and
Hyaluronidase (Wydase.RTM.) Injected in Rabbit Corneas
[0108] Twenty (20) healthy rabbits of the New Zealand cross variety
weighing 1.5 kg to 2.5 kg, were individually marked for
identification and were hosed individually in suspended cages. The
animals received a commercially available pelleted rabbit feed on a
daily basis, with tap water available ad libitum.
[0109] The animals were divided into 4 groups of 5 animals each.
All 20 animals were examined pre-treatment by slit lamp
biomicroscopy and fluorescein staining for pre-treatment health of
the rabbit corneas.
[0110] The following day, each animal was anesthetized by
intravenous administration of a combination of 34 mg/kg of ketamine
hydrochloride and 5-mg/kg xylazine. The eyelids were retracted
using a lid speculum, and the eyes were disinfected with an
iodine-povidone wash.
[0111] Experimental treatments of either balanced salt solution;
Hyaluronidase (Wydase.RTM.) or Hyaluronidase (ACS) was administered
by injection using a 0.3 cc tuberculin syringe with a 29 gauge,
0.5-inch needle attached thereto. The hyaluronidase (ACS) solution
utilized in this example was free of thimerosal and constituted the
specific preferred hyaluronidase ACS formulation set forth in Table
2.
[0112] The experimental treatments administered to each animal
group were as follows:
10 TREATMENT GROUP NO. Right Eye Left Eye 1 BSS Untreated control 2
Hyaluronidase (Wydase) 25 I.U. Hyaluronidase (Wydase) 100 I.U. 3
Hyaluronidase (ACS) 25 I.U. Hyaluronidase (ACS) 100 I.U. 4
Hyaluronidase (ACS) 500 I.U. Hyaluronidase (ACS) 1000 I.U.
[0113] On days 1, 7, 15, and 30 following the injections, the eyes
of the animals were examined grossly and biomicroscopically for
evidence of treatment related toxicities.
[0114] In summary, the eyes of the BSS treated and untreated
control groups were free of toxicity.
[0115] The eyes of Group 2 animals treated with Hyaluronidase
(Wydase.RTM.) preserved with thimerosal were found to be toxic.
[0116] The eyes of Group 3 and Group 4 animals treated with
Hyaluronidase (ACS) were found to be free of toxicity.
[0117] Accordingly, it is concluded that thimerosal containing
Wydase.RTM. formulation does cause toxic effects in the eyes of
rabbits at the dosages tested. However, hyaluronidase (ACS) caused
no toxic effects for these animals at the dosages tested.
[0118] Creation of retinal detachment is an essential component of
macular translocation. Presently retinal detachment is created
using mechanical methods that cause damage to the integrity of the
retina as well as the appearance of Proliferative Vitreoretinopathy
(PVR). Proliferative Vitreoretinopathy is the most common cause of
ultimate failure after surgical treatment for rhegmatogenous
retinal detachment.sup.17, 18. PVR is characterized by epiretinal
and subretinal fibrous proliferation, contraction of membranes,
recurrent retinal detachment, reopening of pre-existing retinal
breaks and formulation of new retinal breaks. Other associated
features of PVR are hypotory, vitreous opacity, aqueous flare, iris
neovascularization and macular pucker.
EXAMPLE 4
[0119] Induction of Reversible Retinal Detachment in Rabbits for
Retinal Translocation using PEG 300 and PEG 400
[0120] In this study, 12-pigmented rabbits were divided into 2
groups of 6 animals each. Group I consisted of six animals that
were injected OD with PEG 300. Each of the animals received were
first injected intravitreally 50.mu.l of 75 I.U. of hyaluronidase
(ACS). Three days after the administration of hyaluronidase, the
animals received 50 .mu.l of PEG 300. As a control, these same
animals received saline injections OS and thus served as a control
group (Group Ia).
[0121] Group II consisted of six animals that were injected that
were injected OD with PEG 400. As with Group I, each of the animals
received were first injected intravitreally 50 .mu.l of 75 I.U. of
hyaluronidase (ACS). Three days after the administration of
hyaluronidase, the animals received 50 .mu.l of PEG 400. As a
control, these same animals received saline injections OS and thus
served as a control group (Group IIa).
[0122] During this procedure a number of ophthalmic methodologies
were utilized to monitor the effects and progress of the various
experimental treatments. For example, indirect ophthalmoscopy was
utilized by a retinal specialist to determine the effects of
administering a retinal detaching dose of the compounds disclosed.
Fundus photography was used to document the effects of the
treatments, in addition to document the retinal detachment as well
as the spontaneous retinal re-attachments. Light microscopy and
electron microscopy of eye samples was used to determine the effect
of the treatments from a histological perspective. Ultrasonography
techniques were used to objectively determine the presence of
retinal detachment. Additionally, electroretinography was performed
to determine changes due to treatment. The observed results of this
experiment are summarized in Table 10.
[0123] The results show that animal injected first with 75 I.U. of
Hyaluronidase enzyme solution followed 3 days later with a second
intravitreal injection of PEG 300 or PEG 400 induced retinal
detachment in the rabbit eyes within 48 hours. The detached retinas
spontaneously re-attached within 3 weeks of the intravitreal
injection of PEG 300 and PEG 400. The control group that received
the sterile saline solution did not produce any retinal
detachments. The intravitreal injection of Hyaluronidase followed
by the intravitreal injection of PEG 300 and PEG 400 was determined
to be safe by using measurement techniques, like Indirect
Ophthalmoscopy, Fundus Photography, Light and Electron Microscopy
and Electroretinography.
[0124] Efficacy of inducing retinal detachments and retinal
re-attachments were documented by Indirect Ophthalmoscopy, Fundus
Photography and Ultrasonography (.beta.-Scan).
[0125] The results in this experiment show that PEG 300 and PEG 400
injected intravitreally are safe and effective in inducing retinal
detachment in 48 hours and within 3 weeks retinal re-attachment
without causing any toxicity.
11TABLE 10 Induction of Reversible Retinal Detachment in Rabbits
for Retinal Translocation using PEG 300 and PEG 400 Retinal
Detachment Retinal Histological and Rabbit at 48 hours Reattachment
Electro Electron Group 1.sup.st Intravitreal 2.sup.nd Intravitreal
Post 2.sup.nd at 3 weeks Retinography Microscopic Number Injection
Injection Injection Post 2.sup.nd Injection Results Results Group I
50 .mu.l of 75 I.U. 50 .mu.l of sterile All All Normal Normal
Treatment of Hyaluronidase PEG 300 Fundus Photos Fundus Photos No
Adverse No Histological n = 6 solution .beta. Scans .beta. Scans
Effects Changes Observed Group Ia 30 .mu.l of 75 I.U. 50 .mu.l of
sterile None Not Applicable Normal Normal Control of Hyaluronidase
saline solution Fundus Photos Fundus Photos n = 6 solution .beta.
Scans .beta. Scan Group II 50 .mu.l of 75 I.U. 50 .mu.l of sterile
All All Normal Normal Treatment of Hyaluronidase PEG 400 Fundus
Photos Fundus Photos No Adverse No Histological n = 6 solution
.beta. Scans .beta. Scans Effects Changes Observed Group IIa 30
.mu.l of 75 I.U. 50 .mu.l of sterile None Not Applicable Normal
Normal Control of Hyaluronidase saline solution Fundus Photos
Fundus Photos n = 6 solution .beta. Scans .beta. Scan
EXAMPLE 5
[0126] Induction of Reversible Retinal Detachment in Rabbits for
Retinal Translocation using Varving Concentration of PEG 300
[0127] In this study, 8-pigmented rabbits were divided into 4
groups of 2 animals each. Group I consisted of 2 animals treated
with 100% PEG 300. As performed in Example 1, these animals were
first injected intravitreally (OD) with 50 .mu.l of 75 I.U. of
Hyaluronidase solution (Group I). Fourteen days after the first
injection these animals received 50 .mu.l of 100% sterile PEG 300
intravitreally. The OS eye was used as the untreated control as
described in Example 1 (Group Ia). A second group of two animals,
Group II, was first injected intravitreally (OD) with 50 .mu.l of
75 I.U. of Hyaluronidase solution and then injected 14 days later
with 50 .mu.l of sterile 75% solution of PEG 300 intravitreally.
The OS eyes of these animals were used as the untreated controls
(Group IIa). Groups II and IIa were treated according to the same
protocols as described for Groups I, Ia, II, and IIa with the
exception that Group III received 50 .mu.l of 50% sterile solution
of PEG 300 intravitreally. Similarly, Groups IV and IVa were
treated according to the same protocols as described above,
however, Group IV received 50 .mu.l of 25% sterile solution of PEG
300 intravitreally.
[0128] All animals were observed at day 1 and 2, weeks 1, 2, 3 and
4 by Indirect Ophthalmoscopy and Fundus Photography. All retinal
detachments and retinal re-attachments were documented using Fundus
Photography and Ultrasonography (.beta.-Scan). In addition, 3
rabbit eyes were enucleated and the eyes were prepared for light
microscopy as well as for Electron Microscopy.
[0129] The observed results in this experiment are summarized in
Table 11. The results show that animals injected with 75 I.U. of
Hyaluronidase enzyme solution followed 14 days later with a second
Intravitreal Injection of varying concentrations of PEG 300 induce
retinal detachment in rabbit eyes within 48 hours. The detached
retinas spontaneously re-attach within 3 weeks of the PEG
injection. The control group does not produce any retinal
detachment.
12TABLE 11 Induction of Reversible Retinal Detachment in Rabbits
for Retinal Translocation using PEG 300 Retinal Detachment Retinal
Rabbit 2.sup.nd At 48 hours Reattachment Electro Histological and
Group 1.sup.st Intravitreal Intravitreal Post 2.sup.nd At 3 weeks
Retinography Electron Microscopic Number Injection Injection
Injection Post 2.sup.nd Injection Results Results Group I - OD 30
.mu.l of 75 I.U. 50 .mu.l of sterile All All No Adverse No Adverse
100% PEG of 100% PEG Fundus Photos Fundus Photos effects were
Histological or Electron 300 Hyaluronidase 300 .beta. Scans .beta.
Scans observed in the Microscopy changes Group II - OD solution 50
.mu.l of sterile Electro- were observed on 3 75% PEG 300 75% PEG
300 retinography years that were Group III- 50 .mu.l of sterile
results after enucleated and prepared OD 50% PEG 300 retinal for
light and Electron 50% PEG 300 detachment and Microscopy. Group IV
- 50 .mu.l of sterile spontaneous OD 25% PEG 300 retinal re- 25%
PEG 300 attachments 3 weeks later. Group Ia - OS None None None Not
Applicable Normal Normal Untreated Fundus Photos Fundus Photos
Control .beta. Scans .beta. Scan Group IIa -OS Untreated Control
Group IIIa - OS Untreated Control Group IVa - OS Untreated
Control
[0130] The intravitreal injection of Hyaluronidase followed the
intravitreal injection of PEG 300 was determined to be safe. The
efficacy of inducing retinal detachment and spontaneous retinal
re-attachment was demonstrated at PEG 300 concentrations of 100%,
75%, 50% and 25% in saline vehicle respectively without causing any
toxicity.
EXAMPLE 6
[0131] Retinal Detachment Reversal in Adult Cats Using
Hyaluronidase
[0132] The study is performed on 15 adult cats (Felis domesticus)
and modeled generally on the study of Mervin, et al., "Limiting
Photoreceptor Death and Deconstruction During Experimental Retinal
Detachment: The Value of Oxygen Supplementation," Am. J. Ophthal.
128:155-164 (1999). Unlike the Mervin study however, the present
experiment examines the effect of hyaluronidase administration to
prevent photoreceptor death rather than the effect of supplemental
oxygen administration.
[0133] Three groups of five adult domestic cats (Experimental Group
I, Experimental Group II, and Control Group I) are anesthetized
using 10 mg/kg of xylazine (Boehringer Ingelheim Vetmedica, Inc.,
St. Joseph, Mo.) and 50 mg/kg of ketamine (Sigma, St. Louis, Mo.).
The surgical method of Anderson, et al., (Invest. Ophthalmol. Vis.
Sci. 27:168-183 (1986)) is used to induce retinal detachment in two
experimental groups of 5 cats each, however, the lens and vitreous
of these cats are left in place during the surgical procedure.
[0134] During the procedure, a glass micropipette is introduced
through a 20-gauge hole in the sclera at the region of the pars
plana. A balanced salt solution (BSS) containing 0.25% sodium
hyaluronate is then infused between the neural retina and retinal
pigment epithelium. A single detachment is produced in the right
eye of each of the 10 cats.
[0135] The experimental groups are allowed to recover for six
hours. The cats of Experimental Group I receive 100 .mu.l of the
PEG formulation described in Table 5 following the recovery period,
while the cats of Experimental Group II and Control Group mH
receive no PEG. The animals are then housed for 3 days at room
temperature with food and water ad libitum and ambient illumination
on a 12-hour/12-hour light/dark cycle with the light phase
consisting of an intensity of approximately 50 lux.
[0136] Following the three day period after detachment surgery and
PEG administration, the animals are sacrificed and the eyes are
enucleated perimortem and immersion fixed for 10 minutes in 4%
paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4. The
cornea and lens are removed and the eyecup is divided into segments
that span the point of detachment.
[0137] The isolated tissue is sectioned on a cryostat or on a
Vibratome (Technical Products International, Warrington, Pa.). For
cryosections, the segments are washed briefly in PBS and then
placed in 15% sucrose until they sink. The pieces are then embedded
and cryosectioned at 20 .mu.m. The tissue destined for Vibratome
sectioning is not dehydrated. After fixation, this tissue is rinsed
in PBS and embedded in 5% agarose in PBS. Sections of 100 .mu.m in
thickness are cut with the Vibratome.
[0138] To detect dying (apoptotic) cells in situ, the terminal
deoxytransferase-mediated dUTP nick end labeling (TUNEL) technique
is used to demonstrate the fragmentation of DNA characteristic of
apoptosis, following the protocol for cryosections using the
fluorescent marker usually Cy3. See Egensperger, et al., Dev. Brain
Res. 97:1-8 (1996). To provide general DNA labeling, the Vibratome
sections are incubated for 4 hours in propidium iodide (0.5
.mu.g/ml in PBS).
[0139] To label cone sheaths by the method of Blanks et al.
(Invest. Ophthalmol. Vis. Sci. 25:546-557 (1984)), the cryosections
and Vibratome sections are incubated in biotinylated peanut
agglutinin (Vector Laboratories, Burlingame, Calif.). The
cryosections are incubated for 1 hour in peanut agglutinin diluted
in PBS to a final dilution of 400 .mu.g per ml, followed by
incubation for 1 hour in streptavidin-Cy1 or Cy3. The Vibratome
sections are incubated overnight in the same solutions.
[0140] For the immunocytochemical study of the cryosections,
incubation times for the blocking serum (10% normal goat serum) and
for the primary and secondary antibodies are 1 to 2 hours, and the
primary antibody is made up in PBS containing Triton X-100 0.3%.
For the Vibratome sections, staining times for the blocking serum
(normal donkey serum, 1:20), primary antibody, and secondary
antibody are 24 hours for each step to allow adequate penetration
into the thick sections. Buffer rinses of 1.5 hours are performed
between all antibody steps. All antibodies and rinse solutions are
made up in PBS containing bovine serum albumin (0.5%) and Triton-X
100 0.1%. On completion of the staining, the sections are mounted
in 5% n-propyl gallate in glycerol. The cryosections and Vibratome
sections are labeled with antibodies to cytochrome oxidase
(Molecular Probes, Eugene, Oreg.) at 1 .mu.g per ml; antibodies to
rod opsin at 1:100; antibodies to blue and red-green cone opsins at
1:1000; to synaptophysin at 1:100; antibodies to glial fibrillary
acidic protein 1:500; antibodies to Ki-67 (the MIB-1 antibody of
Immunotech, Inc., Westbrook, Mass.) at 1:100; antibodies to
.beta.-tubulin at 1:1000; and antibodies to basic fibroblast growth
factor (bFGF) (Upstate Biotechnology, Lake Placid, N.Y.) 1:200
(Valter K., Maslim J., Bowers F., Stone J., Photoreceptor dystrophy
in the RCS rat: roles of oxygen, debris and bFGF, Invest Ophthalmol
Vis Sci 1998;39:2427-2442). The secondary antibodies conjugated to
Cy2 or Cy3 (Jackson ImmunoResearch Laboratories, West Grove, Pa.)
are diluted 1:200 or 1:1000.
[0141] In situ hybridization is performed with cRNA probes prepared
from a 477-bp cDNA strand corresponding to nucleotides 533-1009 of
a rat ovarian bFGF cDNA. This cDNA incorporates the complete bFGF
coding sequence and a 75 nucleotide-3' flanking sequence. The
strand is cloned into pBluescript SK.sup.+ (Stratagene, San Diego,
Calif.) vector. The detailed procedures have been published (Valter
K., Maslim J., Bowers F., Stone J., Photoreceptor dystrophy in the
RCS rat: roles of oxygen, debris and bFGF, Invest Ophthalmol Vis
Sci 1998;39:2427-2442).
[0142] Observations of the thickness of the outer nuclear layer are
made for all the animals after the retinal detachment surgery.
Thinning of the outer nuclear layer is observed in those animals
that receive PEG-300. Cellular effects of detachment on the neural
retina and the retinal pigment epithelium are discussed in: Glaser
B M, editor. Retina: surgical retina, Volume 3, St. Louis: C V
Mosby, 1989:165-190). The numbers of animals studied with each
technique are as follows: TUNEL for two animals kept in room air
and six animals kept in hyperoxia; cone opsin labeling for two and
three animals, respectively; peanut agglutinin labeling, two and
three animals, respectively; synaptophysin labeling, two and six
animals, respectively; cytochrome oxidase labeling, two and five
animals, respectively; and bFGF labeling, two and five animals,
respectively. Images of retinal tissue are digitized by confocal
microscopy. When two fluorophores (red and green) is both
digitized, the images are obtained sequentially to maximize signal
separation. Wherever signal intensities are to be compared, the
photomultiplier tube settings are held constant.
[0143] Molecule-specific signals from immunolabeled proteins are
quantified by use of NIH Image software (the Analysis tool) from
the confocal images. Any optimization of images done before
quantitation was kept identical between images to be compared.
[0144] Results
[0145] Generally, it is found that eyes with detached retinas
showed less degeneration as judged by the histochemical analysis
described above than eyes with detached retinas that did not
receive the PEG composition. More specifically, the detached
retinas in the eyes of the cats not treated with PEG (Group II)
showed signs of greater retinal degeneration than the eyes of the
cats of Group I. These results indicate that the administration of
the compositions described herein is effective in limiting retinal
damage associated with retinal detachments.
EXAMPLE 7
[0146] Reattachment and Damage Protection
[0147] A subject presenting a detached retina is administered
approximately 100 .mu.l of the formulation of Table 5 once a day
for two weeks. The condition of the detached retina is monitored
using standard techniques well known in the art. The administration
of the PEG-300 formulation promotes retinal reattachment and limits
retinal tissue degeneration.
[0148] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
references referred to above are hereby incorporated by
reference.
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